In this work C3bot and peptides derived from thisADP-ribosylating C3
transferase named C3bot156-181 (26 amino acids),C3bot163-177 (15 amino acids)
and before known C3bot154-182 (29 amino acids)were investigated concerning
their effects in different neuronal tissue models. Using immunofluorescence-
based counting of synaptic contactsdetected via antibody-staining of primary
hippocampal neurons an increase ofthe number of dendritic synapses due to the
application of C3bot andC3bot156-181 could be discovered. This could also be
shown by counting contactsstained by using antibodies against VGLUT1/2 and
VGAT. The application ofC3bot163-177 did not have any effect. When growing
neurons on the inhibitorychondroitinesulfate proteoglycans the number of
synaptic contacts wasdecreased. This reduction could be neutralized by C3bot,
C3bot156-181 and thebefore published potent peptide C3bot154-182. A knock-down
of RhoA using thesiRNA RhoA G28 could also raise the number of synaptic
contacts. Combination ofthe siRNA and C3-derivates did not lead to an
additional effect. Analysis of the expression levels of synaptic proteins
inhippocampal cell culture after treatment with C3bot156-181 could show
anincrease of the amount of synaptophysin and SNAP 25. There was no effect
onother pre- und postsynaptic proteins. With organotypical brain slices it is
possible to measurethe length and number of growing axons. C3bot156-181 was
able to influence bothparameters positively with C3bot163-177 having no
effect. Combination of C3botund C3bot156-181 slightly increased the positive
effect. It could be confirmed that the level of activated RhoA ishigher in
injured murine spinal cord. Therefore a pulldown-assay for thedetection of
RhoA-activity was performed and used to detect a possible impactof the
C3-peptides on this level. C3bot156-181, C3bot154-182 as well asC3bot163-177
could reduce the activity of RhoA after seven days. Data on the locomotion of
mice after thoracic spinal cordlesion (hemisection or compression) was added
to the work. C3bot156-181improved the locomotor recovery. The enzyme-deficient
peptides perform their effects viareduction of RhoA-activity. Because of the
minimal concentration of thesubstances during their application a receptor-
dependent mechanism or specificinternalization of the petides can be assumed.
Experiments confirming thisspeculation are in preparation. Seeing the positive
effects of the C3-Peptides onneuroregeneration and their adavantages over
C3bot they appear as potenttherapeutic agents.